Inactivation of light activated reactions is the first step in the recovery of the photoreceptor cell following photoactivation. While our knowledge of activation reactions is rather sophisticated, the mechanisms involved in the inactivation are still uncertain. In the proposed research we will examine the roles of rhodopsin kinase, GTPase and calcium in rod's recovery from photoactivation. In continuing our studies on verifying the rhodopsin kinase hypothesis which contends that phosphorylation inactivates bleached rhodopsin initiating the recovery phase, we will purify the kinase, reconstitute it with the PDE system and examine the kinetics of its phosphorylation of bleached rhodopsin and termination of light activated cGMP hydrolysis. The possible requirement of the 48K protein to enhance the effects of kinase will be investigated. Inactivation of bleached rhodopsin alone will not terminate light activated cGMP hydrolysis unless the G proteins activated by bleached rhodopsin are also inactivated. Activated G protein (G.GTP) is believed to be inactivated by the inherent GTPase activity of G protein, but the kinetics of this reaction as reported in the current literature do not support a role for this activity in the recovery process. We will continue our investigation in testing various factors and experimental conditions influencing the kinetics of the GTPase activity and evaluate if GTPase activity can account for the inactivation of G.GTP. Calcium appears to have strong negative influence on the recovery process. High internal calcium concentration in rod prolongs photoresponse and slows down the recovery. We will investigate the influence of calcium of rhodopsin kinase, GTPase and 48K protein in order to identify the mechanisms by which calcium regulates the recovery process. The nature of the recovery process may not be unique to the visual response. Hormone and neurotransmitter activated systems operate by a receptor-transducer-effector design similar to that in vision and may have similar recovery mechanisms. Progress in our studies in the visual system may therefore have broader applications beyond vision.